title: Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.
abstract: Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.
authors.affiliation: Cell Biology Laboratory, GE Global Research Center, Niskayuna, NY 12309, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gerdes
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Sevinsky
authors.forename: Anup
authors.initials: A
authors.lastname: Sood
authors.forename: Sudeshna
authors.initials: S
authors.lastname: Adak
authors.forename: Musodiq O
authors.initials: MO
authors.lastname: Bello
authors.forename: Alexander
authors.initials: A
authors.lastname: Bordwell
authors.forename: Ali
authors.initials: A
authors.lastname: Can
authors.forename: Alex
authors.initials: A
authors.lastname: Corwin
authors.forename: Sean
authors.initials: S
authors.lastname: Dinn
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Filkins
authors.forename: Denise
authors.initials: D
authors.lastname: Hollman
authors.forename: Vidya
authors.initials: V
authors.lastname: Kamath
authors.forename: Sireesha
authors.initials: S
authors.lastname: Kaanumalle
authors.forename: Kevin
authors.initials: K
authors.lastname: Kenny
authors.forename: Melinda
authors.initials: M
authors.lastname: Larsen
authors.forename: Michael
authors.initials: M
authors.lastname: Lazare
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.forename: Christina
authors.initials: C
authors.lastname: Lowes
authors.forename: Colin C
authors.initials: CC
authors.lastname: McCulloch
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McDonough
authors.forename: Michael C
authors.initials: MC
authors.lastname: Montalto
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Pang
authors.forename: Jens
authors.initials: J
authors.lastname: Rittscher
authors.forename: Alberto
authors.initials: A
authors.lastname: Santamaria-Pang
authors.forename: Brion D
authors.initials: BD
authors.lastname: Sarachan
authors.forename: Maximilian L
authors.initials: ML
authors.lastname: Seel
authors.forename: Antti
authors.initials: A
authors.lastname: Seppo
authors.forename: Kashan
authors.initials: K
authors.lastname: Shaikh
authors.forename: Yunxia
authors.initials: Y
authors.lastname: Sui
authors.forename: Jingyu
authors.initials: J
authors.lastname: Zhang
authors.forename: Fiona
authors.initials: F
authors.lastname: Ginty
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000175:diagnosis*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000175:diagnosis*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011944:Receptors, Androgen / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D018709:Statistics, Nonparametric; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D015100:3,3'-Diaminobenzidine; D014408:Biomarkers, Tumor; D005557:Formaldehyde; D018719:Erb-b2 Receptor Tyrosine Kinases; D011944:Receptors, Androgen; D011960:Receptors, Estrogen; D016159:Tumor Suppressor Protein p53; C508053:ERBB2 protein, human
keywords: cancer diagnostics; high-content cellular analysis; image analysis; mTOR; multiplexing